6-(Bromomethyl)-2,4-diaminopteridine hydrobromide

ABSTRACT

A pteridine compound having the formula of 6-(bromomethyl)-2,4-diaminopteridine hydrobromide. 2,4-diamino-6-pteridine-methanol.HBr is reacted with triphenylphosphine dibromide or phosphorus tribromide to form 6-(bromomethyl)-2,4-diaminopteridine hydrobromide. The bromine atom in the molecule can then be replaced with the functional group, N-[4-(methylamino)-benzoyl]-L-glutamic acid in the case of methotrexate and N-(4-aminobenzoyl)-L-glutamic acid in the case of aminopterin.

This is a Divisional of Application Ser. No. 563,466, filed Mar. 31,1975.

BACKGROUND OF THE INVENTION

This invention relates to a pteridine compound, more particularly,6-(bromomethyl)-2,4-diaminopteridine hydrobromide.

The folic acid antimetabolitesN-[4-[[(2,4-diamino-6-pteridinyl)-methyl]amino]benzoyl]-L-glutamic acid(aminopterin) andN-[4-[[(2,4-diamino-6-pteridinyl)methyl]methylamino]benzoyl]-L-glutamicacid (methotexate) were synthesized nearly 30 years ago. Althoughaminopterin has been used as a folic acid antagonist in the treatment ofleukemia and remains of interest for use as a rodenticide, it has notbecome as important in cancer chemotherapy as methotrexate, which hasbeen in extensive clinical use for over twenty years. Methotrexate hasbecome even more prominent in recent years through its use in newlydeveloped clinical procedures involving its administration in massivedoses followed by treatment with citrovorum factor. The use ofmethotrexate in this manner has greatly increased demands forproduction.

The usefulness of aminopterin and methotrexate as anticancer agentsprompted a still-continuing search for structural variants, analogs, orderivatives that afford greater overall effectiveness. Drawbacks andlimitations in available processes for preparing compounds structurallyrelated to these antimetabolites by the common feature of the(2,4-diamino-6-pteridinyl)methyl grouping (unsubstituted at the7-position) have caused many investigators to seek new syntheticapproaches. The improvements needed in order to increase the attainabletypes and numbers of related compounds are greater versatility,percentage yields, and ease of purification of the products.

SUMMARY OF THE INVENTION

It is, therefore, a primary object of the present invention to provide apteridine compound which improves methods of making pteridine compoundsproviding a high yield, versatility, and ease of purification of theproducts.

It is another object of the present invention to provide a compound formaking methotrexate, aminopterin, and related compounds, which is freeof the aforementioned and other such disadvantages.

The compound according to the present invention achieves these objectsand concomitantly furnishes improved syntheses of aminopterin,methotrexate, and related compounds.

The invention provides for the union of the(2,4-diamino-6-pteridinyl)methyl grouping with diverse side chains. Thisresult is achieved by nucleophilic displacement reactions of2,4-diamino-6-(bromomethyl)pteridine hydrobromide (I) with amino,hydroxyl, and sulfhydryl functions of amines (aromatic and aliphatic),phenols, and thiophenol under appropriate accessary conditions.

Nearly all previously reported work on compounds conceivably or actuallyattainable by the use of I has involved the introduction of the(2,4-diamino-6-pteridinyl)methyl grouping at amine functions. Fourmethods have been used. Each is mentioned briefly below. Comments aregiven on the general synthetic utility of each of the four methods incomparison with that of the new process. Specific evaluative comparisonsare made when possible in terms of methotrexate. Two previously usedmethods of attachment of the (2,4-diamino-6-pteridinyl) methyl groupingto oxygen are described after the background on amino compounds.Apparently, analogous sulfur compounds have not been reported.

Until recently, synthetic approaches used for the preparation ofaminopterin, methotrexate and related compounds were adaptations of amethod used for the preparation ofN-[4-[[(2-amino-4-hydroxy-6-pteridinyl)methyl]amino]benzoyl]-L-glutamicacid (folic acid) generally known as the Waller procedure. Thisprocedure involves the reaction, in one mixture, of three separateorganic components: a 4,5-diaminopyrimidine, a suitable three-carboncompound, and an aromatic amine. Adaptation of the Waller procedure toprepare methotrexate thus employs 2,4,5,6-tetraaminopyrimidine,2,3-dibromopropionaldehyde, and N-[4-(methylamino)benzoyl]-L-glutamicacid. It is our understanding that methotrexate is prepared commerciallyby this procedure. ##STR1## In view of the complexities of the reactionmixtures, it is not surprising that the Waller procedure gives mixturesof products from which pure desired materials are obtained in low yieldsfollowing the use of laborious and tedious techniques. Investigatorsexperienced in the use of the Waller procedure have found that, withsuitable choices of reaction conditions and purification techniques,yields in the range of 5-10% can be obtained. The previously reportedpreparation of methotrexate does not give a percentage yield. Thepreparation of the D-isomer by the Waller procedure was recentlyreported, with N-[4-(methylamino)benzoyl]-D-glutamic acid being usedinstead of the L-form, and a column chromatographic (ion-exchange)procedure devised for the purification of methotrexate as obtainedcommercially was used to advantage to obtain the pure D-form in 6.5%yield. Also in this connection, an elaborate column chromatographic(ion-exchange) method used to separate folic acid analogs has beenadapted for use in an assay procedure for commercial methotrexate inorder to remove impurities prior to spectrophotometric determination ofintact methotrexate. The main contaminant among those removed by thesechromatographic methods is N-[4-[[(2-amino-4-hydroxy-6-pteridinyl)methyl]methyl-amino]benzoyl]-L-glutamic acid (N¹⁰-methylfolic acid). The application of column techniques would beimpractical in large scale commercial production of methotrexate. Aprocess adaptable to commercial production that would affordmethotrexate at acceptable cost, free of N¹⁰ -methylfolic acid, andcontaining lesser amounts of other impurities as compared to presentlyavailable commercial methotrexate would provide obvious advantages. Thepresent process offers those advantages in facile syntheses ofmethotrexate and various analogs.

A recently developed multistep route in which2-amino-3-cyano-5-(chloromethyl)pyrazine 1-oxide serves as the keyintermediate has been used to prepare the pure diethyl ester ofmethotrexate and some analogs with altered side chains. The chloromethylgroup of the key intermediate in this process affords diversity in theattachment of side chains in the same functional manner as thebromomethyl group of I. An important difference, however, is that theuse of I allows direct attachment of the(2,4-diamino-6-pteridinyl)methyl grouping to the side chain while twosteps yet remain after the nucleophilic displacement step using2-amino-3-cyano-5-(chloromethyl)pyrazine 1-oxide. In the sequenceleading to methotrexate diethyl ester shown below, removal of the1-oxide function by treatment with triethyl phosphite was followed byformation of the substituted pteridine ring by condensation of the2-amino-3-cyanopyrazine system with guanidine. ##STR2## The overallyield of pure methotrexate diethyl ester obtained by the above route wascalculated from the reported stepwise yields to be 15%, and columnchromatographic procedures were used at two points in the process. Incontrast, methotrexate itself was obtained in 59% overall yield (for twosteps) after hydrolysis in situ of the diethyl ester prepared directlyfrom I and the same side-chain intermediate by the present method.

Earlier studies on new approaches to methotrexate analogs led to amultistep route to4-[[(2,4-diamino-6-pteridinyl)-methyl]methylamino]benzoic acid. Thisroute is potentially applicable to the synthesis of methotrexate, but itis too lengthy (11 steps) to be considered for use in a synthesisotherwise achievable through use of I.

The only other process of which we are aware that has been used tointroduce the (2,4-diamino-6-pteridinyl)methyl grouping at amino groupsis restricted to the production of secondary amines. It consists ofcondensation of highly unstable 2,4-diamino-6-pteridinecarboxaldehydewith a primary amine under reducing conditions (H₂, PtO₂) to produce asecondary amino group at the point of juncture of the two reactants.Concomitant reduction of the 7,8 double bond of the pteridine ring isreversed by oxidation with iodine. This process is, therefore, notapplicable to the synthesis of methotrexate or any analog derivable bythe procedures mentioned above from a starting compound already bearinga secondary amino group. It has been used to prepare a homolog ofaminopterin in a sequence beginning with the aldehyde and diethylN-[4-(aminomethyl)benzoyl]-L-glutamate as outlined below. ##STR3##

The preparative procedure is scantily described in the published report.Reference is made to the preparation of the corresponding folic acidhomolog, which was purified by paper chromatography. The only advantagethat this approach appears to offer over that of the present inventionis that it affords an unambiguous route to symmetrically substitutedhydrazino types where one of the substituents is the(2,4-diamino-6-pteridinyl) methyl grouping. Obvious disadvantages are(1) the restriction to aminopterin types, (2) the lack of stability ofthe key intermediate, and (3) the attendant complexities that lead toapparently troublesome purification problems.

Little has been published on aminopterin analogs in which the(2,4-diamino-6-pteridinyl)methyl grouping is attached through oxygen toside chains. The 10-oxa analog of aminopterin,N-[α-(2,4-diamino-6-pteridinyl)-4-anisoyl]-L-glutamic acid, andα-(2,4-diamino-6-pteridinyl)-4-anisic acid shown below were obtainedonly as crude products following condensation of2,4,5,6-tetraaminopyrimidine with diethylN-[4-(3,3-diethoxy-2-oxopropoxy)-benzoyl]-L-glutamate and ethyl4-(3,3-diethoxy-2-oxopropoxy)benzoate, respectively. A facilepreparation of the pure 10-oxa analog of aminopterin through appropriateuse of I is described below. ##STR4## From a method viewpoint, theprocess mentioned earlier involving the use of2-amino-3-cyano-5-(chloromethyl)pyrazine 1-oxide could be applied to thesynthesis of the 10-oxa analogs shown above. The only related synthesisthat has actually been done by that method is that of2,4-diamino-6-(methoxymethyl)-pteridine.

Before proceeding with a description of using the instant invention formaking methotrexate, aminopterin, and related compounds, it should benoted that the preparation of I has not heretofore been reported and,therefore, it is still another object of the present invention toprovide a process for the production of6-(bromomethyl)-2,4-diaminopteridine hydrobromide.

According to this aspect of the present invention,2,4-diamino-6-pteridinemethanol obtained from the condensation of2,4,5,6-tetraaminopyrimidine and 1,3-dihydroxyacetone according to areported procedure is converted to its hydrobromide and then treatedwith triphenylphosphine dibromide or phosphorus tribromide.

Generally, the method of making I and the methotrexate, aminopterin, orother related compounds, follows the following scheme.

    __________________________________________________________________________     ##STR5##                                                                     Designation of Y Groupings in Compounds 2-31                                  Compound No.                                                                              Y Grouping*                                                       __________________________________________________________________________    2        N(CH.sub.3)C.sub.6 H.sub.4 CONHCH(CO.sub.2 H)(CH.sub.2).sub.2                 CO.sub.2 H                                                           3        NHC.sub.6 H.sub.4 CONHCH(CO.sub.2 H)(CH.sub.2).sub.2 CO.sub.2 H      4        NHC.sub.6 H.sub.4 CH.sub.2 CONHCH(CO.sub.2 H)(CH.sub.2).sub.2                 CO.sub.2 H                                                           5        NHC.sub.6 H.sub.4 (CH.sub.2).sub.2 CONHCH(CO.sub.2 H)(CH.sub.2).s             ub.2 CO.sub.2 H                                                      6        NHC.sub.6 H.sub.4 SO.sub.2 NHCH(CO.sub.2 H)(CH.sub.2).sub.2                   CO.sub.2 H(as Mg salt)                                                         ##STR6##                                                            8        NHC.sub.6 H.sub.4 CH.sub.2 CONHCH(CO.sub.2 H)CH.sub.2 CO.sub.2                H                                                                    9        NHC.sub.6 H.sub.4 CONH(CH.sub.2).sub.3 CO.sub.2 H                    10       NHC.sub.6 H.sub.4 CONHCH.sub.2 CO.sub.2 H                            11       N(CH.sub.3)C.sub.6 H.sub.4 CO.sub.2 H                                12       NHC.sub.6 H.sub.4 CONH.sub.2                                         13       NHC.sub.6 H.sub.4 CONH(CH.sub.2).sub.2 CH.sub.3                      14       NHC.sub.6 H.sub.4 CON(CH.sub.3).sub.2                                15       NHC.sub.6 H.sub.4 COCH.sub.3                                         16       NHC.sub.6 H.sub.4 NHCOCH.sub.3                                       17       NHC.sub.6 H.sub.4 (CH.sub.2).sub.2 NHCOCH.sub.3                      18       NHC.sub.6 H.sub.4 OCH.sub.3                                          19       NHC.sub.6 H.sub.4 Cl                                                 20       N(CH.sub.3)C.sub.6 H.sub.5                                           21       NHC.sub.6 H.sub.5                                                    22       N(CH.sub.3)(CH.sub.2).sub.4 CONHCH(CO.sub.2 CH.sub.3)(CH.sub.2).s             ub.2 CO.sub.2 CH.sub.3                                               23       N(CH.sub.3)(CH.sub.2).sub.4 CONHCH(CO.sub.2 H)(CH.sub.2).sub.2                CO.sub.2 H                                                           24       NH(CH.sub.2).sub.2 C.sub.6 H.sub.5                                   25       NH(CH.sub.2).sub.3 O(CH.sub.2).sub.2 OCH.sub.2 CH.sub.3              26                                                                                      ##STR7##                                                            27       OC.sub.6 H.sub.4 CONHCH(CO.sub.2 C.sub.2 H.sub.5)(CH.sub.2).sub.2              CO.sub.2 C.sub.2 H.sub.5                                            28       OC.sub.6 H.sub.4 CONHCH(CO.sub.2 H)(CH.sub.2).sub.2 CO.sub.2 H       29       OC.sub.6 H.sub.4 CONH.sub.2                                          30       OC.sub.6 H.sub.5                                                     31       SC.sub.6 H.sub.5                                                     __________________________________________________________________________     *All disubstituted-benzene types are the 1,4-isomeric forms.             

Keeping in mind that the method of making I according to one aspect ofthe present invention has already been described in general terms,according to the second aspect of the present invention, the use of thepteridine compounds according to the invention may be represented asfollows:

The method of making pteridine compounds represented by the formula:##STR8## wherein Y is a member selected from the group consisting of--NRR¹ R², --NH(CH₂)₂ C₆ H₅, ##STR9## --OR³, and --SC₆ H₅ ; R is H orCH₃ ; R¹ is --C₆ H₄ R⁴, ##STR10## or --(CH₂)_(m) --; R² is --H,--CONHCH(COOR⁵)(CH₂)_(n) COOR⁵, --CONHCH₂ COOH, --COOH, --CONH₂,--CONH(CH₂)₂ CH₃, --CON(CH₃)₂, --COCH₃, --NHCOCH₃, --CONH(CH₂)₃ CO₂ H,--(CH₂)₂ NHCOCH₃, --OCH₃, --Cl, or --O(CH₂)₂ OCH₂ CH₃ ;

R³ is C₆ H₄ R⁶ ;

R⁴ is (CH₂)_(p) or (SO₂)_(q) ;

R⁵ is --H or --CH₃ ;

R⁶ is --H, --CONHCH(COOC₂ H₅) (CH₂)₂ COOC₂ H₅, --CONHCH(COOH)(CH₂)₂COOH, or --CONH₂ ;

m is 2, 3 or 4; n is 1 or 2; p is 0, 1 or 2; and q is 0 or 1 comprisingreacting 6-(bromomethyl)-2,4-diaminopteridine hydrobromide with acompound represented by the formula HY, where Y is as defined above, ina reaction medium of N,N-dimethylacetamide or N,N-dimethylformamide andrecovering said pteridine compound.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The starting material, 2,4,5,6-tetraaminopyrimidine was condensed with1,3-dihydroxyacetone according to the procedure of C. M. Baugh and E.Shaw [J. Org. Chem., 29, 3610 (1964)] as described below. The crudematerial obtained directly from the reaction mixture was examined byproton magnetic resonance (pmr) spectroscopy in solution indeuteriotrifluoroacetic acid (CF₃ CO₂ D) and found to be predominantlythe desired 2,4-diamino-6-pteridinemethanol as evidenced by signals atδ5.3 (CH₂) and δ9.1 (pteridine position-7). Relatively weak signals nearδ2.8 (CH₃) and δ8.8 (pteridine ring H) in the spectrum produced by thecrude product indicated the presence of one or possibly both of the 6-or 7-methyl-substituted 2,4-diaminopteridines. The hydroxymethylcompound was obtained in nearly pure form after conversion of themixture of products to hydrobromide salts. The greater solubility of thecontaminants in this form in ethanol allowed their nearly completeremoval from the hydroxymethyl compound. Pure2,4-diamino-6-methylpteridine hydrobromide was isolated from anethanolic filtrate and identified as the 6-methyl isomer by comparisonsof its uv-absorption spectra with those of an authentic sample, but noclear evidence was obtained that proved the presence of the 7-methylisomer. A small percentage of methyl-substituted contaminant thatremained in the hydroxymethyl intermediate persisted after theconversion to I (see details below), but was not detectable by pmrspectroscopy or thin-layer chromatography (tlc) in any of the productsprepared from I.

The hydroxymethyl compound was converted to I by two procedures. Thepreferred procedure (Method A) makes use of triphenylphosphine dibromidein N,N-dimethylacetamide (DMAC). In Method B, phosphorus tribromide inN,N-dimethylformamide (DMF) was successfully used, but the yield wasmuch lower than that afforded by Method A.

Samples of I have been stored in refrigerators in tightly sealedcontainers protected from light for several months without evidentdeterioration. Gradual darkening has been observed in samples exposedfor several weeks to ordinary lighting and ambient conditions in thelaboratory.

In all but two of the preparations from I given below, anhydrous DMACwas used as the reaction medium. In the exceptions (6 and 16), whichwere not tried in DMAC, hexamethylphosphoric triamide was used, probablyto no advantage.

In the reaction of I with HY, the ratio of reactants can vary widely,but the preferred molar ratio of I to HY is from 1:1 to 1:4.

Melting points, or approximate decomposition points, are given for someof the compounds described below. They were observed on either aMel-Temp apparatus or a Kofler Heizbank as indicated in the procedure.In general, these substituted pteridines, particularly those isolated ashydrohalides, lack meaningful melting points. They usually decompose ata high temperature without melting, and the temperature at whichdecomposition begins is difficult to determine and reproduce. For thatreason, melting point determinations were not attempted on all of thecompounds prepared. The pmr spectra were determined with a VarianXL-100-15 spectrometer in the solvent indicated (CF₃ CO₂ D or deuterateddimethyl sulfoxide, DMSO-d₆) using tetramethylsilane as internalreference. Chemical shifts quoted for multiplets were measured from theapproximate centers, and relative integrals of signal areas are givenfor the assignment indicated. The uv-absorption spectra were determinedwith a Cary Model 14 spectrophotometer. Unless indicated otherwise,thinlayer chromatograms on all compounds in which the side chain bears aterminal carboxyl group were run on DEAE-cellulose plates using 0.5 Msodium chloride, 0.2 M in mercaptoethanol, in 0.005 M potassiumphosphate buffer at pH 7.0 and were viewed by uv lamps emitting at 254nm and 365 nm.

EXAMPLE 1. 2,4-Diamino-6-pteridinemethanol Hydrobromide.

2,4,5,6-Tetraaminopyrimidine.H₂ SO₄.H₂ O (75.0 g, 0.293 mole) was addedto a stirred solution of BaCl₂.2H₂ O (71.5 g, 0.293 mole) in H₂ O (1.45l.) at 85°-90°. The mixture was stirred rapidly at ˜90° for 15 min,cooled to 40°, and filtered from BaSO₄, which was washed thoroughly on afunnel with H₂ O. The clear, yellow filtrate was then diluted furtherwith H₂ O to give a volume of 4.35 l. This solution of thetetraaminopyrimidine.2HCl was then added to a solution of NaOAc (4.35 l.of 4 N) in which dihydroxyacetone (79.3 g, 0.88 mole) andcysteine.HCl.H₂ O (51.5 g, 0.293 mole) had just been dissolved. Theresulting solution was stirred mechanically at room temperature while aslow stream of air was continuously passed through it for 26 hr.(Yellow-orange solid began separating after 2 hr.) The mixture was thenkept in a refrigerator for 16 hr before the solid was collected, washedsuccessively with cold H₂ O, EtOH, and Et₂ O before it was dried toconstant weight in vacuo over P₂ O₅ at 25°. [The crude product mixture(47 g) was weighed in order to obtain an estimate of the volume of 48%HBr required to form hydrobromide salts.] A mechanically stirred mixtureof the dried solid and EtOH (6.05 l.) was heated to 70°, and a solutionof 48% HBr (28 ml) in EtOH (490 ml) was added in a thin stream while themixture was maintained at 70°-75°. The mixture was then refluxed forabout 5 min with rapid stirring while nearly all of the solid dissolved.The hot solution was treated with Norit and filtered through a Celitemat. The clear yellow filtrate was kept in a refrigerator overnightwhile a first crop of orange-colored solid separated. The collectedsolid was washed with EtOH, then dried in vacuo (56° over P₂ O₅) to give17.2 g of product. The filtrate was concentrated by evaporation (rotaryevaporator, H₂ O aspirator, bath to 35°) to about 2 l. and thenrefrigerated to give a second crop, which was dried as before, of 10.2g; total yield 27.4 g (34%). The pmr spectrum of this material in CF₃CO.sub. 2 D showed it to contain a barely detectable amount ofmethylsubstituted 2,4-diaminopteridine.HBr as evidenced by very weaksignals at δ2.83 (CH₃) and δ8.85 (pteridine ring H). Strong signalsproduced by the desired product occur at δ5.28 (6-CH₂ O) and δ9.08 (C₇-H). The proportion of desired product to the methyl-substitutedcontaminant was estimated from the pmr integrals to be 20:1. The pmrspectrum also revealed retention of a small amount of EtOH in theproduct dried as described but not enough to interfere with theconversion of it to I.

EXAMPLE 1A 2,4-Diamino-6-(bromomethyl)pteridine Hydrobromide (I)

Method A.

Bromine (59.6 g, 0.373 mole) was added dropwise over a 30-min period toa stirred solution of triphenylphosphine (97.7 g, 0.373 mole) inanhydrous DMAC (486 ml) kept at ˜10° (ice bath) and protected fromatmospheric moisture. (Bromine remaining in the funnel was rinsed with10 ml of DMAC). A smooth suspension containing finely divided,crystalline triphenylphosphine dibromide resulted. The2,4-diamino-6-pteridinemethanol.HRr (25.4 g, 0.093 mole) described abovewas added in one portion through a powder funnel (with the aid of 10 mlDMAC). The ice bath was removed, and the stirred mixture was allowed towarm to 20°-25°. After about 1 hr, complete solution had occurred. Thesolution, which gradually developed a dark-red color, was kept at20°-25° for 1 hr longer and was then chilled (ice bath) before it wastreated with EtOH (72 ml). After overnight refrigeration, the solventswere removed by evaporation in vacuo (Swissco evaporator, pressure <1mm, bath <45°). The dark, semisolid residue was stirred with two 300-mlportions of C₆ H₆ (to remove triphenylphosphine oxide), and each portionwas removed from the C₆ H₆ -insoluble product by decantation. The solidthat remained was dissolved with stirring in glacial AcOH (660 ml) whichhad been preheated to 80°. The mixture was kept in a bath at 80° untilsolution was complete Tan crystalline solid separated as the darksolution was allowed to cool. Overnight refrigeration caused the AcOH topartially freeze. When it had thawed, the solid was collected, washedwith chilled AcOH followed by Et₂ O, and dried in vacuo (over P₂ O₅ andNaOH pellets) at successive temperatures of 25°, 56°, and 110°. (Thehigher temperature was necessary for complete removal of AcOH). Theyield was 15.3 g (49%). (Some runs afforded 60% yield). This sample wasfurther purified by reprecipitation from MeOH solution (Norit) byaddition of Et₂ O followed by drying in vacuo (25°, P₂ O₅), yield 13.0 g(42%) of pale-yellow solid. Spectral data: λmax, nm (ε x 10⁻³), 0.1 NHCl, 249 (17.3), 339 (10.5), 353 (sh); pH 7, 258 (21.2), 370 (6.87); 0.1N NaOH, 258 (21.5), 370 (6.94); pmr (CF₃ CO₂ D), δ 4.70 (s, 2, CH₂) and9.08 (s, 1, C₇ -H); estimated proportion relative to themethyl-substituted contaminant, 25:1. These spectral properties are inclose agreement with those of a similarly obtained sample that gave thefollowing elemental analysis results. Anal. Calcd for C₇ H₇ BrN₆.HBr: C,25.02; H, 2.40; Br, 47.56; N, 25.01. Found: C, 25.22; H, 2.44; Br,47.30; N, 24.99. The 13.0-g sample described above gave the followingresults. Anal. Found: C, 25.59; H, 2.79; N, 24.62. The preparation of Idescribed above is typical of several runs that gave similar yields ofmaterial whose pmr spectra differed only slightly in the estimatedproportion of I with respect to the methyl-substituted contaminant. Theproportions usually ranged from 16:1 to 25:1, which corresponds to apercentage of I of 94 to 96%. Samples of I of this degree of purityproved to be suitable for use in the preparation of 2-31.

EXAMPLE 1B

(i) method B.

Anhydrous DMF (50 ml) was added in one portion with rapid stirring tofreshly distilled PBr₃ (5.0 g, 18 mmoles). A mildly exothermic reactionoccurred with the temperature of the resulting mixture rising to 35°within 2 min. The stirred mixture was allowed to cool to 28° while awhite solid precipitated. 2,4-diamino-6-pteridinemethanol hydrobromide(5.0 g, 18 mmoles) was then added. The temperature of the stirredmixture rose rapidly to 37°, and the solids dissolved. Stirring wascontinued for 30 min while the temperature returned to 25°. Et₂ O (200ml) was then added with stirring while dark semisolid materialseparated. The mixture was stirred for 1 hr before the supernatant wasremoved by decantation. The residue was stirred with more Et₂ O, whichwas also removed by decantation, and was then dissolved in AcOH (40 ml).The dark solution was left overnight while crude solid separated. Thebrown solid was collected, washed successively with AcOH and Et₂ O, andthen dissolved in CH₃ OH (40 ml). Norit treatment (for about 3 min)followed by filtration through Celite gave a clear yellow solution,which was diluted with Et₂ O to precipitate I as a paleyellow solid in16% yield (1.0 g). Examination of this material by tlc (silica gel, 4:1CHCl₃ -CH₃ OH) showed one major spot, which fluoresced under uv light,with an impurity remaining at the origin a second reprecipitation fromNorit-treated CH₃ OH solution by addition of Et₂ O removed most of theimmobile contaminant but lowered the yield to 11% (0.68g); pmr (CF₃ CO₂D)δ4.7 (s, 2, CH₂), 9.1(s,1,C₇ -H) and a weak spurious signal at δ4.0(CF₃ CO₂ CH₃, from retained CH₃ OH after esterification with thesolvent). The proportion of I to CH₃ OH of 4:1 estimated from the pmrspectrum is consistent with the results of elemental analysis. Anal.Calcd for C₇ H₇ BrN₆.HBr.O.25CH₃ OH; C, 25.31; H, 2.64; N, 24.43. Found:C, 25.14; H, 2.84; N, 24.18.

EXAMPLE 2.N-[4-[[(2,4-Diamino-6-pteridinyl)methyl]methylamino]-benzoyl]-L-glutamicacid (2, Methotrexate) Trihydrate.

A stirred mixture of I (0.34 g, 1.0 mmole) and diethylN-[4-(methylamino)benzoyl]-L-glutamate (0.37 g, 1.1 mmole) in DMAC (4ml) was kept at 53°-57° (bath temperature) for 4 hrs. (Solution occurreda few minutes after heating was started.) The dark-orange solution wascooled to room temperature before H₂ O (20 ml) and NaOH solution (2 mlof 2 N) were added successively with rapid stirring (no externalcooling). The finely divided orange-yellow precipitate that formeddissolved readily with stirring. When solution occurred more H₂ O (10ml) was added. After 16 hrs at room temperature the orange solution wastreated with Norit (about 50 mg) and filtered through a Celite mat. Themat was washed with H₂ O until the washings were colorless. The combinedfiltrate and wash solution was treated with sufficient 1 N HCl to lowerthe pH to 5.5. The turbid mixture was then clarified by treatment withNorit followed by filtration through Celite as before. The filtrate (nowapproximately 85 ml volume) was treated with 1 N HCl to lower the pH to4.0 where a voluminous yellow-orange precipitate formed. The mixture wasrefrigerated for about 2 hrs before the precipitate was collected anddried in vacuo (25°-30°, P₂ O₅). The dried solid (0.32 g) was dissolvedin NaOH solution (20 ml of 0.08 N). After treatment with Norit andfiltration as before, the pH was lowered (from 12.1) to 5.5. The faintlyturbid mixture was clarified (Norit, Celite) as before, and the clearfiltrate was acidified to pH 4.0. After a refrigeration period, theyellow-orange solid was collected, washed with H₂ O, and dried in vacuo(25°-30°, P₂ O₅) to give 2.3H₂ O in 59% yield (0.30 g). Anal. Calcd forC₂₀ H₂₂ N₈ O₅.3H₂ O: C, 47.24; H, 5.55; N, 22.04. Found: C, 47.42; H,5.15; N, 22.05. Spectral data: λmax, nm (ε×10⁻³). 0.1 N HCl, 243 (18.9),307 (22.4); pH 7, 257 (24.8), 302 (25.1), 370 (7.90); 0.1 N NaOH, 257(25.6), 302 (25.1), 370 (8.14); pmr (DMSO-d₆), δ2.05 (m, 2, CHCH₂ CH₂),2.30 (m, 2, CH₂ CO₂ H), 3.20 (s, 3, CH₃), 4.38 (m, 1, NHCHCO₂ H), 4.82(s, 2, CH₂ N), 6.85 and 7.73 (m, 4, C₆ H₄), 7.05 (broad s, 2, NH₂), 7.9(very broad s, 2, NH₂), 8.20 (d, 1, NHCO), 8.62 (s, 1, C₇ -H); ir,identical with that of an authentic sample. A thin-layer chromatogramrevealed one uv-absorbing spot (identical with that produced byauthentic 2) with a very faint fluorescent spot at or near the origin.The appearance of this chromatogram agrees with the publisheddescription of that produced by the D-form of 2 prepared by the Wallerprocedure and purified by a method that included ion-exchange columnchromatography.

The results of an independent analytical examination show thatmethotrexate prepared by the simple procedure given above is obtained ina better state of purity than present USP methotrexate.

EXAMPLE 3.N-[4-[[(2,4-Diamino-6-pteridinyl)methyl]amino]benzoyl]-L-glutamic Acid(3, Aminopterin) Hydrate (4:7).

A mixture of I (168 mg, 0.500 mmole) and N-(4-aminobenzoyl)-L-glutamicacid (400 mg, 1.50 mmoles) in DMAC (2 ml) was stirred at 25° under N₂ ina stoppered flask protected from light. Solution occurred after 2 hrs.After 18 hrs, the orange solution was mixed with H₂ O (15 ml) withstirring to give a finely divided, yellow precipitate. The mixture wascentrifuged, and the supernatant removed by decantation. The yellowsolid was stirred with four 15-ml portions of H₂ O, each of which wasremoved by decantation after centrifugation. The solid was thensuspended in EtOH (15-20 ml), collected by filtration, washed with Et₂O, and dried in vacuo (25°, P₂ O₅) to give hydrated 3 in 68% yield (160mg). Anal. Calcd for C₁₉ H₂₀ N₈ O₅.1.75H₂ O: C, 48.36; H, 5.02; N,23.74. Found: C, 48.72; H, 4.91; N, 23.36. Spectral data: λ max. nm (ε ×10⁻³), 0.1 N HCl, 244 (18.2), 290 (20.5), 335 (11.0); pH 7, 260 (26.7),283 (25.5), 370 (8.00); 0.1 N NaOH, 260 (26.9), 283 (25.3), 370 (8.00);pmr (DMSO-d₆), δ2.02 (m, 2, CHCH₂ CH₂), 2.32 (m, 2, CH₂ CO₂ H), 4.36 (m,1, NHCHCO₂ H), 4.52 (s, 2, CH₂ N), 6.85 (m, 4, 2 phenylene protons plusNH₂), 7.72 (m, 2, phenylene), 7.86 (broad s, 2, NH₂), 8.13 (d, 1, NHCO),8.72 (s, 1, C₇ -H). Examination by tlc revealed one uv-absorbing spotand no fluorescence at any point. The uv data given above is inagreement with reported results.

EXAMPLE 4N-[4-[[(2,4-Diamino-6-pteridinyl)methyl]amino]phenylacetyl]-L-glutamicAcid (4) Monohydrate.

Treatment of N-(4-aminophenylacetyl)-L-glutamic acid (5.60 g, 20.0mmoles) with I (2.24 g, 6.67 mmoles) in DMAC (25 ml) as described forthe preparation of 3 was followed by dropwise addition of thedark-orange reaction solution to stirred H₂ O (250 ml). Orange solidseparated, and the mixture was stirred at 25° for 30 min and then atabout 5° for 1 hr before the precipitate was collected by filtration.The suction-dried solid was removed from the funnel and stirred with H₂0 (125 ml). This mixture was centrifuged, and the clear supernatant wasremoved by decantation. After a second wash with H₂ O followed bycenrifugation and decantation, the residue was again suspended in H₂ Oand collected by filtration. The suction-dried solid was then washedwith Et₂ O before it was dried in vacuo (25° over P₂ O₅); yield 72%(2.27 g). Anal. Calcd for C₂₀ H₂₂ N₈ O₅.H.sub. 2 O: C, 50.84; H, 5.12;N, 23.72. Found: C, 51.00; H, 4.90; N, 24.02. Spectral data: λ max, nm(ε × 10³¹ 3), 0.1 N HCl, 246 (18.3), 337 (10.1); pH 7, 256 (31.1), 370(7.52); 0.1 N NaOH, 256 (31.8), 370 (7.57); pmr (DMSO-d₆), δ 1.90 (m, 2,CHCH₂ CH₂), 2.28 (m, 2, CH₂ CO₂ H), 3.32 (s, 2, CH₂ CON), 4.22 (m, 1,NHCHCO₂ H), 4.44 (s, 2, CH₂ N), 6.66 (m, 4, 2 phenylene protons plusNH₂), 7.02 (m, 2, phenylene), 7.32 (s, 1, CH₂ NH), 7.84 (broad s, 2,NH₂), 8.15 (d, 1, NHCO), 8.72 (s, 1, C.sub. 7 -H). Tlc revealed oneuv-absorbing spot with a very thin and faintly fluorescent cap.

EXAMPLE 5.N-[4-[[(2,4-Diamino-6-pteridinyl)methyl]amino]hydrocinnamoyl]-L-glutamicAcid (5) Dihydrate.

A mixture of I (2.24 g, 6.67 mmoles) and N-(4-aminohydrocinnamoyl)L-glutamic acid (20.0 mmoles) in DMAC (25 ml) was stirred at 25° underN₂ in a stoppered flask protected from light. The mixture graduallythinned but separation of a precipitate commenced just before solutionhad occurred. After 24 hrs, the mixture was poured into H₂ 0 (250 ml) togive a red-orange precipitate, which was readily collected byfiltration, washed on the funnel with several portions of H₂ O followedby Et₂ O, and dried in vacuo (25° over P₂ O₅); yield 72% (2.44 g). Tlcrevealed a fluorescent spot above the dark, uv-absorbing spot and alsoat the origin. The crude product was then stirred with H₂ O (80 ml), and0.1 N NaOH (100 ml) was added with stirring. The dark-red solution thatformed was treated with Norit, filtered (Celite), chilled to about 5°,and treated with 0.1 N HCl (100 ml). The orange precipitate that formedwas collected by filtration, washed on the funnel with H₂ O followed byEt₂ O, and dried in vacuo (25° over P₂ O₅); yield 38% (1.27 g.). Anal.Calcd for C₂₁ H₂₄ N₈ O₅. 2H₂ O: C, 50.00; H, 5.59; N, 22.21. Found: C,50.38; H, 5.00; N, 22.42. (A sample obtained from a trial run in themanner and percentage yield stated above gave the following elementalanalysis results. Anal. Found: C, 50.37, 50.40; H, 5.29, 5.12; N, 22.63,22.37). Spectral data: λ max, nm (ε × 10⁻³), 0.1 N HCl, 246 (19.4), 336(10.8); pH 7, 256 (30.9), 370 (7.80); 0.1 N NaOH, 256 (31.3), 370(7.76); pmr (DMSO-d₆), δ1.88 (m, 2, CHCH₂ CH₂), 2.1-2.8 (overlappingmultiplets, 6, C₆ H₄ CH₂ CH₂ CO and CH₂ CO₂ H), 4.22 (m, 1, NHCHCO₂ H),4.42 (s, 2, CH₂ N), 6.65 (m, 4, 2 phenylene protons plus NH₂), 6.69 (m,2, phenylene), 7.80 (broad s, 2, NH₂), 8.05 (d, 1, NHCO), 8.70 (s, 1, C₇-H). Tlc showed one uv-absorbing spot with a thin, faintly fluorescentcap and a barely discernible fluorescent spot at the origin.

EXAMPLE 6.N-[4-[[(2,4-Diamino-6-pteridinyl)methyl]amino]benzenesulfonyl]-L-glutamicAcid (6), Magnesium Salt, Heptahydrate.

A solution of I (2.63 g, 7.82 mmoles) andN-(4-aminobenzenesulfonyl-L-glutamic acid (2.60 g, 8.60 mmoles) inhexamethylphosphoric triamide (50 ml) was kept at about 25° for 6 daysand then added to H₂ O (150 ml) with stirring. The mixture wasrefrigerated for about 4 hrs before it was centrifuged. The darkgel-like precipitate was washed with H₂ O (three times with 25-30 mlportions) until the clear supernatant following centrifugation becamepale-yellow (as opposed to dark-orange initially). The precipitate,still as dark hydrous gel, was stirred with H₂ O (120 ml) and treatedwith NaOH solution (2ml of 4 N). The dark-orange solution that formedwas treated with Norit and filtered (Celite mat) to give a pale-orangefiltrate which was treated with glacial AcOH to produce pH 4.0. A yellowgel-like precipitate formed. Following overnight refrigeration, themixture was centrifuged and the residue was washed once with H₂ O asbefore and then Me₂ CO. Following decantation after centrifugation fromthe Me₂ CO wash, the residue was again suspended in Me₂ CO, collected byfiltration, washed with Me₂ CO followed by Et₂ O, and dried in vacuo(25°, P₂ O₅) to give crude 6 as a brown solid (1.29). This material wastreated in boiling H₂ O (100 ml) with MgO (500 mg) with rapid stirring,and, after about 5 min all the brown solid had dissolved. The hotmixture was clarified (Norit, Celite,) and the clear yellow filtrate wasrefrigerated overnight while the Mg salt of 6 separated as a yellowsolid. The mixture was centrifuged, and the Mg salt was washed once withcold H₂ O (20 ml). After centrifugation, the wash solution and the firstdecantate were combined and set aside for further processing. The solidwas then suspended in Me₂ CO, collected by filtration, washed with Et₂O, and air dried to constant weight (0.95 g) before it wasrecrystallized from the minimum volume of hot H₂ O (about 30 ml) to givethe pure Mg salt as pale-yellow lustrous crystals, which were collectedas before and dried in vacuo (25°, P₂ O₅). The sample was then allowedto equilibrate with ambient conditions of the laboratory whereupon itunderwent a weight increase (from 0.60 g to 0.76 g) that corresponds totransformation of the anhydrous salt to a heptahydrate. Elementalanalysis results agree with that indication. The yield was 16%. Anal.Calcd for C₁₈ H₁₈ MgN₈ O₆ S.7H₂ O: C, 34.60; H, 5.16; Mg, 3.89; N,17.93. Found: C, 34.28; H, 5.24; Mg, 3.64; N, 17.67. Spectral data:λmax, nm (ε × 10⁻³), 0.1 N HCl, 245 (17.3), 272 (25.0), 337 (10.6); pH7, 266 (33.5), 370 (7.82); 0.1 N NaOH, 263 (35.4), 372 (8.04); pmr (CF₃CO₂ D), δ 2.25 (m, 2, CHCH₂ CH₂), 2.73 (m, 2, CH₂ CO₂ H), 4.36 (m, 1,NHCHCO₂ H), 5.30 (s, 2, CH₂ N), 7.85 and 8.18 (m, 4, C₆ H₄), 9.02 (s, 1,C₇ -H). The sample was homogeneous according to tlc (one uv-absorbingspot).

The filtrate from the recrystallization step and the decantate from thecentrifugation step were combined, and the pH of the solution waslowered by addition of AcOH from 8.5 to 4.0 to give the acid 6 as apale-yellow solid (0.45 g). Examination of this product by tlc with thepure Mg salt as reference showed the acid to contain impurities thatproduced pale fluorescent streaks above and below the expecteduv-absorbing spot. Further purification of the free acid was not pursuedsince the pure Mg salt was suitable for testing purposes.

EXAMPLE 7. N-[[5- [[(2,4-Diamino-6-pteridinyl)methyl]amino]-2-pyridinyl]-carbonyl]-L-qlutamic acid (7,2'-Azaaminopterin) Monohydrate.

Diethyl N-[(5-amino-2-pyridinyl)carbonyl]-L-glutamate (3.076 g, 9.5mmoles) and I (3.19 g, 9.5 mmoles) were dissolved in dry DMAC (40 ml).The flask was flushed with N₂ and sealed, and the reaction mixture wasstirred at about 25° for 7 days in the dark. The DMAC was removed byevaporation in vacuo, and the residue was dissolved in a solutionconsisting of H₂ O (230 ml), EtOH (200 ml), and NaOH solution (48 ml of1 N). This solution was kept in a stoppered flask under N₂ at about 25°for 6 hrs. The solution was then neutralized to pH 7, and the EtOH wasevaporated in vacuo. The volume of the aqueous solution remaining wasincreased to 500 ml, and the precipitate obtained by acidification to pH4 was isolated and washed with H₂ O by centrifugation. The wet cake wasfreeze-dried to a brown solid (3.85 g). A solution (pH 8) of 3.2 g ofthis material in dilute KOH solution (200 ml) was treated with Norit andthen filtered through Celite. The filtrate was neutralized to pH 7, andthe Norit treatment was repeated. The resulting filtrate was acidifiedto pH 4, and the precipitate was isolated and washed with H₂ O bycentrifugation. Treatment of the wet cake with Me₂ CO afforded a solid(1.64 g) which was isolated by filtration. This solid was purified bycolumn chromatography using DEAE-cellulose (Mannex Regular Low Capacity;column dimensions 4.2 × 51 cm) prepared in the following manner. The dryDEAE-cellulose (150 gm) was hydrated, and the fines were removed so thatabout one-half of the original DEAE-cellulose remained. This materialwas deaerated, poured into the column and then washed successively withpotassium phosphate buffer of pH 7.0 (3 l.), H₂ O (10 l.), and 0.2 Maqueous 2-mercaptoethanol (2 l.). The crude product was dissolved indilute aqueous KOH (1.5 l.) which was 0.2 M in 2-mercaptoethanol, andthis solution (pH 6.5) was applied to the column. The column was washedwith 0.2 M aqueous 2-mercaptoethanol (1 l.) and then eluted using astepwise gradient (0.1-0.3 M) of NaCl solutions of pH 7.0, 0.2 M in2-mercaptoethanol and 0.005 M in potassium phosphate buffer. The uvabsorbance of the column eluate was monitored continuously at 300 nm.The product moved down the column as a yellow-orange zone, and theproduct-containing fractions were divided into four groups according totheir uv absorbance. Each fraction group was acidified (1 N HCl) to pH 4and refrigerated. The individual precipitates were isolated bycentrifugation and redissolved in H₂ O (200 ml) containing2-mercaptoethanol (2 ml) by the addition of 1 N KOH to pH 8. Thesesolutions were filtered, and the filtrates were acidified to pH 4 andrefrigerated. The yellow precipitates were isolated and washed with H₂ O(four times) by centrifugation. The supernatant solutions were retainedfor lyophilization, and the wet precipitates were freeze-dried to yellowamorphous solids. On the basis of tlc analysis, three of these solidswere combined (220 mg). The fourth solid and the solid obtained bylyophilization of the combined supernatant washes were redissolved indilute KOH solution as before, and these solutions (pH 7) were treatedwith Norit and filtered. Acidification of the filtrates, and isolationand freeze-drying of the precipitates as before afforded an additional178 mg of product. The total yield was 11% (398 mg). Anal. Calcd for C₁₈H₁₉ N₉ O₅.H₂ O: C, 47.06; H, 4.61; N, 27.44. Found: C, 47.08; H, 4.58;N, 27.51. Spectral data: λmax, nm (ε × 10⁻³), 0.1 N HCl, 223 (22.3). 242(21.6), 290 (19.2), 342 (18.9); pH 7, 259 (24.6), 282 (21.4), 305 (sh),370 (7.94); 0.1 N NaOH, 259 (24.7), 282 (21.1 ), 305 (sh), 370 (8.13);pmr (CF₃ CO₂ D), δ 2.00-2.67 (overlapping multiplets, 4, CH₂ CH₂), 5.00(s, 2, CH₂ N), 5.07 (m, 1, NHCHCO₂ H), 7.90 (doublet of doublets, 1,C_(5') -H), 8.43 (d, 1, C_(6') -H), 8.47 (d, 1, C_(3') -H), 9.05 (s, 1,C₇ -H).

EXAMPLE 8.N-[[4-[[(2,4-Diamino-6-pteridinyl)methyl]amino]phenyl]-acetyl]-L-asparticAcid (8) Hemihydrate.

N-[(4-Aminophenyl)acetyl]-L-aspartic acid (2.34 g, 8.8 mmoles) and I(2.69 g, 8 mmoles) were dissolved in DMAC (40 ml). The flask was flushedwith N₂ and closed, and the reaction mixture was stirred at about 25°for 4 days in the dark. The reaction mixture was filtered, and the solidon the funnel was washed with DMAC. The filtrate was treated with H₂ O(250 ml), and the suspended product was dissolved by addition of theminimum of 1 N NaOH. The solution (pH 7.5) was treated with Norit andfiltered through Celite. The filtrate was acidified to pH 6.2, and themixture was clarified (Norit, Celite) as before. Acidification of thefiltrate to pH 4 afforded an orange precipitate, which was isolated bycentrifugation and then redissolved as before by addition of 1 N NaOH asrequired to a suspension in H₂ O (250 ml). This solution was acidifiedto pH 6.3 and clarified (Norit, Celite) once more. The filtrate wasacidified to pH 3.9 and refrigerated. The orange precipitate wasisolated and washed with cold H₂ O (four times) by centrifugation. Thewet product was lyophilized, pulverized, and dried further in vacuo overP₂ O₅ for 24 hrs; yield 37% (1.3 g). Anal. Calcd for C₁₉ H₂₀ N₈ O₅.O.5H₂O: C, 50.78; H, 4.71; N, 24.93. Found: C. 50.69; H, 4.72; N, 25.09.Spectral data: λmax, nm (ε × 10⁻³), 0.1 N HCl, 246 (19.6), 291 (5.69),337 (10.3), 349 (sh); pH 7, 257 (31.7), 370 (7.49); 0.1 N NaOH, 257(32.4), 370 (7.92); pmr (CF₃ CO₂ D), δ 3.27 (m, 2, CH₂ CO₂ H), 3.93 (s,2, C₆ H₄ CH₂ CO), 5.17 (m, 1, CHNH), 5.23 (s, 2, CH₂ NH); 7.60 (s, 4, C₆H₄), 9.0 (s, 1, C₇ -H).

EXAMPLE 9.4-[N-[4-[[(2,4-diamino-6-pteridinyl)methyl]amino]benzoyl]-amino]-butyricAcid (9) Hydrobromide Hydrate with N,N-Dimethyl-acetamide (20:20:12:5).

A suspension of 4-(4-nitrobenzamido)-butyric acid (1.12 g, 4.75 mmoles)in H₂ O (55 ml) was hydrogenated in the presence of a 5%palladium-on-charcoal catalyst at room temperature and atmosphericpressure. The catalyst was removed by filtration, and the filtrate wasevaporated to dryness in vacuo. The resulting residue of4-(4-aminobenzamido)-butyric acid (1.02 g) was dissolved in DMAC (10 ml)containing I (504 mg, 1.50 mmoles), and the whole was stirred at about25° for 60 hrs. The precipitate of the product was collected byfiltration, washed with Et₂ O and dried in vacuo (78°, P₂ O₅) for 68hrs; yield 63% (483 mg). This sample decomposed from about 240°(Mel-Temp) and was homogeneous by tlc (5:1 CHCl₃ -MeOH). Anal. Calcd forC₁₈ H₂₀ N₈ O₃.HBr.O.60H₂ O.O.25 DMAC: C, 44.75; H, 4.83; N, 22.66.Found: C, 44.54; H, 4.81; N, 22.34. Spectral data: λ_(max), nm (ε ×10⁻³), 0.1 N HCl, 244 (16.6 ); 287 (16.9), 337 (9.58); pH 7, 261 (24.4),279 (22.4), 370 (6.82); 0.1 N NaOH, 260 (24.7), 279 (22.6), 370 (7.06);pmr (DMSO-d₆), δ 1.72 (m, 2, CH₂ CH₂ CH₂), 2.25 (m, 2, CH₂ CO₂ H), 3.23(m, 2, CONHCH₂), 4.63 (s, 2, CH₂ N), 7.22 (m, 4, C₆ H₄), 8.09 (m, 1,NHCO), 8.86 (s, C₇ -H), 9.34 (NH). The pmr spectrum also showed thepresence of DMAC; the NH₂ and CO₂ H groups were too broad to locate butare observed in the integral.

EXAMPLE 10.N-[4-[[(2,4-Diamino-6-pteridinyl)methyl]amino]benzoyl]-glycine (10)Sesquihydrate.

A solution of I (1.01 g, 3.00 mmoles) and N-(4-amino-benzoyl)glycine(640 mg, 3.30 mmoles) in DMAC (10 ml) was stirred under N₂ in astoppered flask protected from light for 4 days. The yellow precipitatethat formed was collected by filtration, washed with DMAC (twice with4-ml portions), suspended in H₂ O (200 ml), and dissolved by addition ofthe required volume of 1 N KOH. The solution (pH 11) was treated withNorit and filtered (Celite mat). The filtrate was brought to 400 mlvolume, acidified to pH 4 by addition of 1 N HBr, and refrigerated. Theyellow precipitate that formed was isolated and washed (four times) withH₂ O by centrifugation. The solid was finally suspended in Me₂ CO (350ml), collected by filtration, and dried in vacuo; yield 55% (604 mg).Anal. Calcd for C₁₆ H₁₆ N₈ O₃.1.5H₂ O: C, 48.60; H, 4.84; N, 28.34.Found: C, 48.82; H, 4.85; N, 28.40. Spectral data: λ_(max), nm (ε ×10⁻³), 0.1 H HCl, 244 (18.0), 288 (20.7), 336 (11.2), 348 (sh); pH 7,260 (27.5), 282 (25.4), 371 (8.20); 0.1 N NaOH, 260 (27.1), 282 (25.4),371 (8.20); pmr (CF₃ CO₂ D), δ 4.50 (s, 2, CH₂ CO₂ H), 5.34 (s, 2, CH₂N), 8.00 (m, 4, C₆ H₄), 9.04 (s, 1, C₇ -H).

EXAMPLE 11. 4-[[(2,4-Diamino-6-pteridinyl)methyl]methylamino]benzoicAcid (11) Sesquihydrate.

A solution of I (168 mg, 0.500 mmole) and 4-(methylamino)benzoic acid(83 mg, 0.55 mmole) in DMAC (2 ml) was stirred at about 25° for 114 hrsand then mixed with H₂ O (18 ml) to cause separation of 11. Thecollected and dried solid (150 mg) was reprecipitated from Norit-treatedand filtered (Celite) NaOH solution (7.5 ml of 0.08 N) by treatment with1 N HCl to produce pH 6.5. The yellow solid was collected, washed withH₂ O, and dried in vacuo (78° over P₂ O₅); yield 60% (105 mg). Anal.Calcd for C₁₅ H₁₅ N₇ O₂.1.5H₂ O: C, 51.13; H, 5.15; N, 27.83. Found: C,51.02; H, 5.24; N, 27.52. Spectral data: λ_(max), nm (ε × 10⁻³), 0.1 NHCl, 240 (17.7), 311 (25.9), 350 (sh) (9.83); pH 7, 258 (25.4), 285(22.9), 372 (7.21); 0.1 N NaOH, 258 (25.8), 285 (22.9), 372 (7.48); pmr(DMSO-d₆), δ 3.23 (s, 3, CH₃ N), 4.82 (s, 2, CH₂), 6.74 (s, 2, NH₂),6.84 and 7.76 (m, 4, C₆ H₄), 7.58 (very broad s, 2, NH₂), 8.60 (s, 1, C₇-H). The uv and pmr spectral data listed are in good agreement withreported data for a sample that gave satisfactory elemental analysisresults for 11.0.65 HCl, and, surprisingly, the ir spectrum of thatsample is identical with that of 11.1.5 H₂ O described above.Examination of 11.1.5H₂ O by tlc revealed one uv-absorbing spot with abarely discernible fluorescent spot just above it.

EXAMPLE 12. 4-[[2,4-Diamino-6-pteridinyl]methyl]aminobenzamide (12)Hydrobromide Monohydrate.

A solution of I (500 mg, 1.49 mmoles) and 4-aminobenzamide (410 mg, 2.98mmoles) in DMAC (20 ml) was stirred at about 25° for 20 hrs. The depositof bright yellow solid was collected by filtration, washed with coldDMAC, and dried in vacuo (25°, P₂ O₅); yield 330 mg. Evaporation of thefiltrate gave a solid residue that was washed thoroughly with Et₂ O andEtOH; yield 220 mg. The two crops produced identical thin-layerchromatograms and were combined; total yield 94% (550 mg). Treatment ofa finely ground suspension of this solid with 0.5 M NaHCO₃ solutionresulted in incomplete removal of HBr. The recovered solid (0.40 g) wasconverted to the full hydrobromide salt by suspending it in EtOH (20ml), adding 48% HBr (0.10 ml), and diluting slowly with Et₂ O (200 ml).The yellow solid was dried in vacuo (78°, P₂ O₅); yield 62% (380 mg); mpchars, but does not melt below 350° (Mel-Temp). Anal. Calcd for C₁₄ H₁₄N₈ O.HBr.H₂ O; C, 41.09; H, 4.19; N, 27.38; Br, 19.52. Found: C, 41.23;H, 3.69; N, 27.44; Br, 19.73. Spectral data: λ_(max), nm (ε × 10⁻³), 0.1N HCl, 243 (17.5), 289 (19.2), 336 (11.2); pH 7, 259 (25.0), 284 (21.6),371 (7.9); 0.1 N NaOH, 259 (25.6). 284 (21.6). 371 (8.2); pmr (DMSO-d₆),δ 4.59 (s, 2, CH₂ N), 7.22 (m, C₆ H₄ and NH₂), 8.81 (m, 3, NH₂ and C₇-H).

EXAMPLE 13. 4-[[(2,4-Diamino-6-pteridinyl)methyl]amino]-N-propylbenzamide (13) Hydrobromide.

A solution of 4-nitro-N-propylbenzamide (1.00 g, 4.80 mmoles) in MeOH(25 ml) was hydrogenated in the presence of a 5% palladium-on-charcoalcatalyst at about 25° and atmospheric pressure. The catalyst was removedby filtration, and the filtrate was evaporated to dryness in vacuo togive 4-amino-N-propylbenzamide as a gummy residue; yiel 836 mg. Thisproduct was dissolved in DMAC (10 ml) containing I (504 mg, 1.50mmoles), and the whole was stirred at room temperature for 72 hrs. Theprecipitate of the product was collected by filtration, washed with Et₂O and dried in vacuo over P₂ O₅ ; yield 74% (482 mg), mp > 340°(Mel-Temp). This sample was homogeneous on tlc (5:1 CHCl₃ --MeOH). Anal.Calcd for C₁₇ H₂₀ N₈ O.HBr: C, 47.12; H, 4.88; N, 25.86. Found: C,47.10; H, 5.18; N, 25.55. Spectral data: λ_(max), nm (ε × 10⁻³), 0.1 NHCl, 244 (18.5), 284 (18.3), 337 (10.5), 345 (sh) (9.70); pH 7, 261(26.9). 279 (24.2), 371 (7.65); 0.1 N NaOH, 261 (27.1), 279 (24.2), 371(7.73); β 0.96 (t, 3, CH₃), 1.50 (m, 2, CH₂ CH₃), 3.17 (m, 2, NHCH₂CH₂), 4.62 (s, 2, CH₂ N), 7.23 (m, 4, C₆ H₄), 8.07 (m, 1, NHCO), 8.87(s, 1, C₇ -H), 9.35 (NH). Some of the NH₂ peaks were too broad to locatebut are observed in the integral.

EXAMPLE 14.4-[[2,4-Diamino-6-pteridinyl]methyl]amino-N,N-dimethylbenzamide (14)Hydrobromide Hydrate.

A solution of I (500 mg, 1.49 mmoles) and 4-amino-N,N-dimethylbenzamide(740 mg, 4.47 mmoles) in DMAC (20 ml) was stirred at about 25° for 48hrs. The solution, which had become turbid after about 24 hrs, wasevaporated to dryness in vacuo. The yellow residue was trituratedthoroughly with several portions of Et₂ O. The finely pulverized solidwas stirred with H₂ O (40 ml), and the mixture was adjusted to pH 7.5with 1 N NaOH. The insoluble solid was collected by filtration andwashed with H₂ O by centrifugation. Elemental analysis results showedthat the material obtained had been only partially converted to the freebase. The product obtained in 56% yield (330 mg) underwent gradualdecomposition without melting above 220° (Mel-Temp). Anal. Calcd for C₁₆H₁₈ N₈ O.O.52 HBr.H₂ O: C, 48.23; H, 5.19; Br, 10.43; N, 28.12. Found:C, 48.40; H, 5.07; Br, 10.47; N, 28.28. Spectral data: λ_(max), nm (ε ×10⁻³), 0.1 N HCl, 245 (21.3), 275 (12.8), 336 (10.7); pH 7, 261 (30.9),372 (7.7); 0.1 N NaOH, 260 (32.1), 372 (7.9); pmr (DMSO-d₆), δ 2.94 (s,6, CH₃), 4.42 (broad overlapping multiplets, CH₂, NH, H₂ O), 7.01 (m, C₆H₄, NH₂), 8.51 (s, 2, NH₂), 8.78 (s, 1, C₇ -H).

EXAMPLE 15. 6-[(4-Acetylphenylamino)methyl]-2,4-pteridinediamine (15)Hydrobromide.

A magnetically stirred mixture of I (1.01 g, 3.00 mmoles) and4-aminoacetophenone (1.62 g, 12.0 mmoles) in DMAC (20 ml) in acentrifuge tube became clear within 15 min, and 15.HBr began separatingafter 30 min. The mixture was stirred 20 hrs at 25° (stoppered under N₂and protected from light) before it was centrifuged. Sucessive washes ofthe orange solid with DMAC (5 ml) and EtOH (three times with 15-mlportions) were each followed by centrifugation and decantation. Theresidue was finally stirred with Et₂ O, collected by filtration, anddried in vacuo (25°, P₂ O₅); yield 68% (0.80 g). Anal. Calcd for C₁₅ H₁₅N₇ O.HBr: C, 46.17; H, 4.13; N, 25.12. Found: C, 46.45; H, 4.29; N,25.38. Spectral data: λ_(max), nm (κ × 10⁻³), 0.1 N HCl, 241 (20.3), 327(27.8); pH 7, 258 (22.0), 327 (24.1); 0.1 N NaOH, 258 (22.1), 327(24.1); pmr (CF₃ CO₂ D), δ 2.84 (s, 3, CH₃), 5.36 (s, 2, CH₂ N), 7.89and 8.33 (m, 4, C₆ H₄), 9.08 (s, 1, C₇ -H),

EXAMPLE 16. N-[4-[(2,4-Diamino-6-pteridinyl)methylamino]phenyl]acetamide(16) Hydrate.

A mixture of I (336 mg, 1.00 mmole) and 4'-aminoacetanilide (451 mg,3.00 mmoles) in hexamethylphosphoric triamide (6 ml) was stirred for 22hrs and poured into H₂ O (25 ml) containing 1 N NaOH (2.0 ml). Theresulting mixture was stirred in an ice bath and the orange precipitatecollected, washed successively with H₂ O, 4:1 Et₂ O-MeOH and Et₂ O anddried in vacuo (100°, P₂ O₅); yield 69% (240 mg), mp > 260° (KoflerHeizbank). Anal. Calcd for C₁₅ H₁₆ N₈ O.1.2H₂ O: C, 52.08; H, 5.36; N,32.39. Found: C, 51.99; H, 4.94; N, 32.38. Spectral data: λ_(max), nm (ε× 10⁻³), 0.1 NHCl, 246 (30.8), 336 (10.9), 350 (sh) (9.52); pH 7, 259(33.0), 372 (7.62); 0.1 N NaOH, 259 (34.1), 373 (7.81); pmr (DMSO-d₆), δ1.96 (s, 3, COCH₃), 4.42 (d, 2, CH₂ N), 6.12 (s, 1, NHCH₂), 6.57, 7.72(d, NH₂), 6.66 and 7.32 (m, 4, C₆ H₄), 8.71 (s, 1, C₇ -H), 9.52 (s, 1,NHCO).

EXAMPLE 17.N-[2-[4-[[(2,4-diamino-6-pteridinyl)methyl]amino]phenyl]ethyl]acetamide(17) Hydrobromide Dihydrate.

A solution of N-[2-(4-aminophenyl)ethyl]acetamide (713 mg, 4 mmoles) andI (672 mg, 2 mmoles) in DMAC (8 ml) was stirred under N₂ at about 25° ina stoppered flask protected from light for 2 days. The precipitate wasisolated by filtration and the solid on the funnel was washed with DMAC(2 × 4 ml). The filtrate was set aside, and the solid was then washedwith EtOH (5 ml) and H₂ O (2 × 3 ml). The addition of an equal volume ofH₂ O to the DMAC filtrate afforded more product which was isolated byfiltration and washed with H₂ O. Evaporation of the EtOH-H₂ O washesalso afforded more product, which was triturated with Et₂ O and isolatedby filtration. The combined yield was 70% (609 mg). A sample obtained inthis manner was recrystallized from MeOH to give the pure product inabout 50% recovery. Anal. Calcd for C₁₇ H₂₀ N₈ O.HBr.2H₂ O: C, 43.50; H,5.37; N, 23.88. Found: C, 43.40; H, 5.42; N, 23.82. Spectral data:λ_(max), nm (ε × 10⁻³), 0.1 N HCl, 246 (17.9), 292 (5.80), 337 (10.2),347 (sh); pH 7, 257 (27.6), 280 (sh), 372 (7.70); 0.1 N NaOH, 257(28.6), 280 (sh), 372 (7.96); pmr (DMSO-d₆), δ 1.76 (s, 3, CH₃), 2.56(m, 2, C₆ H₄ -CH₂ CH.sub. 2), 3.16 (m, 2, CH₂ NHCO), 4.52 (s, 2, CH₂NHC₆ H₄), 6.80 (m, 4, C₆ H₄), 8.84 (s, 1, C₇ -H).

EXAMPLE 18. 6-[[(4-Methoxyphenyl)amino]methyl]-2,4-teridinediamine (18)Hydrobromide.

A solution of I (2.69 g, 8.00 mmoles) and freshly recrystallized4-methoxybenzeneamine (1.97 g, 16.0 mmoles) in DMAC (26 ml) was stirredunder N₂ at about 25° in a stoppered flask protected from light for 3days. The product that separated was collected under N₂ in subduedlight, and washed with DMAC, H₂ O, and EtOH. Subsequent operations werealso carried out in subdued light and under N₂ whenever possible. Thesolid was dried in vacuo (25°, P₂ O₅) to give crude 18.HBr in 56% yield(1.68 g). Recrystallization of part (1.28 g) of this material from MeOHled to two crops of brick-red 18.HBr (555 and 219 mg). The secondsmaller crop was slightly less pure than the first, which gave thefollowing analytical results. Anal. Calcd for C₁₄ H₁₅ N₇ O.HBr.O.1CH₃OH: C, 44.40; H, 4.33; N, 25.70. Found: C, 44.59; H, 4.12; N, 25.94.Spectral data: λ_(max), nm (ε × 10⁻³), 0.1 N HCl, 246 (19.0), 337(10.6), 348 (sh); pH 7, 257 (26.6), 280 (sh), 3.72 (7.70); 0.1 N NaOH,257 (27.1), 280 (sh), 372 (7.80); pmr (DMSO-d₆), δ3.17 (s, CH₃ OHsolvate), 3.64 (s, 3, CH₃ O), 4.50 (s, 2, CH₂ N), 6.73 (m, 4, C₆ H₄),8.86 (s, 1, C₇ -H).

EXAMPLE 19. 6-[(4-Chloroanilino)methyl]-2,4-pteridinediamine (19).

Compound I (336 mg, 1.00 mmole) was added to a solution of4-chloroaniline (383 mg, 3.00 mmoles) in DMAC (5 ml), and the resultingmixture was stirred under N₂ for 17 hrs and poured into H₂ O (25 ml).The yellow precipitate of crude hydrobromide was collected byfiltration, washed with H₂ O, then Et₂ O and dried in vacuo (P₂ O₅). Asuspension of the hydrobromide (339 mg) in H₂ O (30 ml) containing 1 NNaOH (1.80 ml) was stirred for 3 hrs. The yellow product was collected,washed with H₂ O, then Et₂ O, and dried at 100° in vacuo (P₂ O₅); yield85% (255 mg), mp > 360° (Kofler Heizbank). Anal. Calcd for C₁₃ H₁₂ CIN₇: C, 51.75; H, 4.01; N, 32.49. Found: C, 51.65; H, 4.15; N, 32.78.Spectral data: λ_(max), nm (ε × 10⁻³), 0.1 N HCl, 246 (21.3), 290 (sh)(5.81), 237 (10.5), 350 (sh) (9.16); pH 7, 257 (33.1), 371 (7.86); 0.1 NNaOH (unstable); pmr (DMSO-d₆), δ 4.44 (d, 2, CH₂), 6.46 (m, NH), 6.60,7.73 (d, NH₂), 6.73 and 7.14 (m, 4, C₆ H₄), 8.72 (s, 1, C₇ -H).

EXAMPLE 20. 6-[N-Methylanilino)methyl]-2,4-pteridinediamine (20).

A solution of I (500 mg, 1.49 mmoles) and excess freshly distilledN-methylaniline (5 ml) in DMAC (20 ml) was stirred at about 25° for 48hrs. The yellow solid that precipitated was collected by filtration,washed with cold DMAC and dried in vacuo (25°, P₂ O₅); yield 26% (110mg). The solid was stirred for a few minutes with cold 0.5 M NaHCO₃solution (25 ml), collected by filtration, and washed by centrifugation.Recrystallization from hot EtOH (50 ml) gave a fluorescent yellowpowder; yield 7% (30 mg), mp 266°-268° dec. (Mel-Temp). Anal. Calcd forC₁₄ H₁₅ N₇ : C, 59.77; H, 5.38; N, 34.85. Found: C, 59.99; H, 5.47; N,34.64. Spectral data: λ_(max), nm (ε × 10⁻³), 0.1 N HCl, 247 (15.5), 290(sh), 337 (13.6), 350 (sh); pH 7, 257 (26.7), 371 (6.32); 0.1 N NaOH,257 (27.2), 371 (6.61); pmr (DMSO-d₆), δ 3.10 (s, 3, CH₃), 4.68 (s, 2,CH₂ N), 6.88 (m, 7, NH₂ and C₆ H₅), 7.54 (s, 2, NH₂), 8.53 (s, 1, C₇-H).

The filtrate from the 110 mg crop was evaporated to give a yellowresidue which was washed thoroughly with Et₂ O and EtOH and then dried;yield 0.34 g. Treatment with NaHCO₃ solution and recrystallization fromEtOH as described above gave additional 20, mp 258°-260° dec (Mel-Temp),in 57% yield (240 mg). Anal. Calcd for C₁₄ H₁₅ N₇ : C, 59.77; H, 5.38;N, 34.85. Found: C, 59.66; H, 5.30; N, 34.67.

EXAMPLE 21. 6-[(Phenylamino)methyl]-2,4-pteridinediamine (21)Hydrobromide.

Solid I (1.01 g, 3.00 mmoles) was added in 4 equal portions during 35min (at intervals of sufficient time to allow the preceding portion todissolve) to a stirred solution of freshly distilled aniline (1.11 g,12.0 mmoles) and DMAC (20 ml) at 20°-25°. A clear solution formed soonafter the last addition, and then, after about 5 min, crystallineproduct began separating. The mixture was stirred 22 hrs longer with thereaction flask purged with N₂, stoppered, and protected from light. Thecollected yellow precipitate of 21. HBr was washed successively withcold H₂ O, Me₂ CO, and Et₂ O; yield 79% (0.82 g). Recrystallization fromH₂ O (˜250 ml required) afforded pure 21.HBr in 38% yield (0.40 g).Anal. Calcd for C₁₃ H₁₃ N₇.HBr: C, 44.84; H, 4.05; N, 28.16. Found: C,45.09; H, 4.16; N, 27.96. Spectral data: λ_(max), nm (ε × 10⁻³), 0.1 NHCl, 246 (18.4), 292-303 (plateau) (5.35), 336 (10.0), 350 (sh) (8.80);pH 7, 257 (27.2), 372 (7.24); 0.1 N NaOH, 257 (27.4), 372 (7.55); pmr(CF₃ CO₂ D), ε 5.28 (s, 2, CH₂), 7.6 (m, 5, C₆ H₅), 9.02 (s, 1, C₇ -H).

EXAMPLE 22.Dimethyl-N-[5-[[(2,4-diamino-6-pteridinyl)methyl]methylamino]-pentanoyl]-L-glutamate(22) Hemihydrate.

KOC₄ H₉ -t (6.55g, 58.4 mmoles) was added to a chilled solutioncontaining I (4.91 g, 14.6 mmoles) and diethylN-[5-(methylamino)pentanoyl]-L-glutamate hydrobromide (11.6 g, 29.2mmoles) in DMAC (150 ml). The mixture was stirred at about 25° under N₂in a closed flask protected from light for 19 hrs. The reaction mixturewas filtered to remove insoluble matter, and the filtrate was evaporatedin vacuo. The gummy residue was dissolved in MeOH (20 ml), and thesolution was filtered and diluted with CHCl₃ (80 ml). This solution wasapplied to a silica gel column (450 g of Brinkmann's Silica Gel H, Type60), and the column was eluted with CHCl₃ :MeOH (4:1). According to tlcresults all fractions that contained product contained also someunreacted starting ester. These fractions were combined and evaporated.The semisolid residue was stirred with EtOH (20 ml), and the yellowsolid that formed was collected. Addition of Et₂ O (200 ml) to thefiltrate gave more yellow solid, which was collected and washed with alittle EtOH. The pmr and mass spectra of these two crops showed each tobe the hydrobromide of the expected product; pmr (DMSO-d₆), δ 2.70 (s,3, CH₃ N), 4.05 (pair of quartets, 4, OCH₂), 1.17 (t, 6, OCH₂ CH₃); m/e490 (M⁺). An unsuccessful attempt was made to dissolve this material inwarm MeOH for column chromatography as before. The MeOH was evaporatedin vacuo, and the yellow residue was suspended in EtOH (50ml). KOC₄ H₉-t was added in portions until solution occurred. The KBr formed wasremoved by filtration, and the filtrate was evaporated. The residue wasdissolved in CHCl₃ -MeOH (4:1, 50 ml), and the solution was applied to asilica gel column (120 g of Brinkmann's Silica Gel H, Type 60). Thecolumn was eluted with 4:1 CHCl₃ -MeOH, the tic-homogeneous fractionswere combined and evaporated in vacuo. The yellow solid that remainedwas triturated with Et₂ O and dried in vacuo (25°, P₂ O₅); yield 12%(816 mg) of product which had undergone transesterification to thecorresponding dimethyl ester. Anal. Calcd for C₂₀ H₃₀ N₈ O₅.O.5H O: C,50.95; H, 6.63; N, 23.76. Found: C, 50.97; H, 6.43; N, 23.91. Spectraldata: pmr (DMSO-d₆), δ 1.26-2.44 [overlapping multiplets, 12, (CH₂)₄ COand (CH₂)₂ CO], 2.16 (s, 3, CH₃ N), 3.56 (s, 3, CH₃ O), 3.60 (s, 3, CH₃O), 3.64 (s, 2, CH₂ NCH₃), 4.27 (m, 1, NHCHCO), 8.18 (d, 1, CONH), 8.73(s, 1, C₇ -H); mass spectrum, m/e 462 (M.sup. +).

EXAMPLE 23.N-[5-[[(2,4-diamino-6-pteridinyl)methyl]methylamino]pentanoyl]-L-glutamicAcid (23) Dihydrochloride Sesquihydrate.

A solution of 22 (542 mg, 1.15 mmoles) in deaerated 0.1 N NaOH (92 ml)was stirred in the dark at about 25° in a closed flask under N₂ for 4hrs. The solution was treated with 1 N HCl to produce pH 6 and dilutedto 1 l. This solution was applied to a DEAE-cellulose column (4 × 25 cm;prepared from Mannex Regular Low Capacity DEAE-Cellulose by de-finingand deaerating) which had been treated with 0.5 M NaCl (1 l.), 0.5 H NCl(1 l.), 0.5 N NH₄ OH (1 l.), and H₂ O (6 l.). The uv absorbance of thecolumn eluate was continuously monitored at 250 nm during application,washing, and elution. After application was complete, the column waswashed with H₂ O (1.2 l.). The product was eluted with 0.02 N HCl, andits travel on the column could be observed by means of its bluefluorescence in uv light. The product-containing fractions, which werestrongly uv absorbing, were pooled and lyophilized. The solid thusobtained was dissolved in 50 ml of water, and this solution was filteredand lyophilized. The product was faintly yellow, very light and fluffy;yield 89% (549 mg). Anal. Calcd for C₁₈ H₂₆ N₈ O₅.2HCl. 1.5H₂ O: C,40.46; H, 5.85; N, 20.97; Cl, 13.27. Found: C, 40.49; H, 5.56; N, 20.80;Cl, 13.54. Spectral data: λ_(max), nm (ε × 10⁻³), 0.1 N HCl 246 (16.6),297 (6.50), 337 (10.0), 348 (sh); pH 7, 225 (10.7), 262 (24.1), 373(7.30); 0.1 N NaOH, 225 (12.1), 258 (24.3), 369 (7.50); pmr (DMSO-d₆), δ1.4-2.4 [overlapping multiplets, 10, (CH₂)₃ CO and (CH₂)₂ CO], 2.78 (s,3, CH₃ N), 3.18 (m, 2, NCH₂ CH₂), 4.20 (m, 1, NHCHCO), 4.69 (s, 2,PterCH₂ NCH₃), 8.22 (d, 1, CONH), 8.96 (s, 1, C₇ -H).

EXAMPLE 24. 6-[(Phenethylamino)methyl]-2,4-Pteridinediamine (24).

Compound I (336 mg. 1.00 mmole) was added to a stirred solution ofphenethylamine (970 mg, 8.00 mmoles) in DMAC (5 ml) at 0°, and theresulting suspension was stirred at 25° for 18 hrs and poured into H₂ O(25 ml). The crude product was collected by filtration, washed with H₂O, dried in vacuo (100°, P₂ O₅) and redissolved in DMAC (8 ml). Thesolution was diluted with H₂ O (1 ml), stirred for 10 min, filtered andevaporated to dryness in vacuo. The residue of yellow solid wastriturated with Et₂ O and dried in vacuo (100°, P₂ O₅); yield 59% (173mg), mp about 219° dec (Kofler Heizbank). Anal. Calcd for C₁₅ H₁₇ N₇ :C, 61.00; H, 5.80; N, 33.20. Found: C, 60.98; H, 5.84; N, 33.10.Spectral data: λ_(max), nm (ε × 10⁻³), 0.1 N HCl, 246 (16.4), 293 (sh)(5.23), 337 (9.70), 350 (sh) (8.5); pH 7, 225 (11.5), 261 (23.4), 373(7.13); 0.1 N NaOH, 258 (23.4), 371 (7.09); pmr (DMSO-d₆), δ 2.78 (s, 4,CH₂ CH₂), 3.90 (s, 2, PterCH₂ N), 6.57, 7.29 (d, 4, NH₂), 7.23 (s, 5, C₆H₅), 8.72 (s, 1, C₇ -H).

EXAMPLE 25. 6-[[3-(2-Ethoxyethoxy)propylamino]methyl]-2,4-pteridinediamine (25).

A mixture of I (336 mg, 1.00 mmole) and 3-(2-ethoxyethoxy)propylamine(588 mg, 4.00 mmoles) in DMAC (5 ml) was stirred for 18 hrs, filtered,and evaporated to dryness in vacuo. A solution of the residue in MeOH(10 ml) was filtered and applied to two Brinkmann Silica Gel F-254preparative thinlayer chromatography plates and developed with MeOH. Theband of yellow product was extracted with hot MeOH and the extractevaporated to a solid which was triturated with Et₂ O (2 ml) and driedin vacuo (100°, P₂ O₅); yield 29% (95 mg), mp about 207° dec (KoflerHeizbank). Anal. Calcd for C₁₄ H₂₃ N₇ O₂.sup.. O.2H₂ O: C, 51.74; H,7.26; N, 30.17. Found: C, 51.70; H, 7.34; N, 30.25. Spectral data:λ_(max), nm (ε × 10⁻³), 0.1 N HCl, 245 (16.1), 290 (sh) (5.18), 336(9.75), 350 (sh) (8.46); pH 7, 225 (11.5), 261 (23.0), 373 (7.17); 0.1 NNaOH, 225 (12.2), 257 (23.3), 370 (7.34); pmr (DMSO-d₆), δ 1.07 (m, 3,CH₃), 1.67 (m, 2, CH₂ CH₂ CH₂), 2.59 (m, NCH₂ CH₂), 3.40-3.64 (m, CH₂O), 3.85 (s, 2, PterCH₂ N), 6.55, 7.60 (d, 4, NH₂), 8.74 (s, 1, C₇ -H).

EXAMPLE 26. 6-[(Cyclohexylamino)methyl]-2,4-pteridinediamine (26)Hemihydrate.

Solid I (2.77 g, 8.30 mmoles) was added in 4 equal portions during 1 hrto a stirred solution of cyclohexaneamine (2.45 g, 24.8 mmoles) in DMAC(33 ml). The mixture was stirred 21 hrs at 25° with the reaction flaskpurged with N₂, stoppered, and protected from light. H₂ O (400 ml) wasadded to the stirred reaction mixture, and the solution (pH 9.7) quicklydeposited a yellow solid which was removed by filtration after 10 min. Asmall additional amount of solid was removed after 15 min longer. Thesetwo solids were discarded. The solution was now flushed thoroughly withN₂, and the pH of the solution was adjusted to 12 by the addition of 2 NNaOH. Gradual separation of yellow solid occurred during refrigerationfor 45 min. The solid was collected, and the filtrate deposited moreyellow precipitate over a period of one hour while N₂ was passed throughthe solution. The second crop of yellow solid was also collected. Bothcrops were washed with water and Et₂ O before being dried in vacuo (25°,P₂ O₅); total yield 46% (1.03 g). Anal. Calcd for C₁₃ H₁₉ N₇.sup..0.5.H₂ O: C, 55.30; H, 7.14; N, 34.73. Found: C, 55.18; H, 7.14; N,34.88. Spectral data: λ_(max), nm (ε × 10⁻³), 0.1 N HCl, 245 (17.9), 382(9.15), 336 (10.4), 347 (sh); pH 7, 261 (24.0), 372 (7.10); 0.1 N NaOH,257 (23.9), 371 (7.27); pmr (DMSO-d₆), δ0.8-2.0 (m, 10, cyclohexaneCH₂), 2.40 (m, 1, NCH), 3.88 (s, 2, CH₂ N), 6.52 (s, 2, NH₂), 7.59 (s,2, NH₂), 8.74 (s, 1, C₇ -H).

EXAMPLE 27. DiethylN-[α-(2,4-diamino-6-pteridinyl)-4-anisoyl]-L-glutamate (27) Hemihydrate.

A mixtue of NaH (0.30 g of 50% dispersion in oil, 6.2 mmoles) anddiethyl N-(4-hydroxybenzoyl)-L-glutamate (2.00 g, 6.20 mmoles) in DMAC(25 ml) was stirred under N₂ with ice-bath cooling unit until solutionwas complete and H₂ evolution had ceased. Solid I (1.04 g, 3.10 mmoles)was then added. The resulting dark-red solution was kept at about 25°under N₂ in a stoppered flask protected from light for 8 days. Additionto dilute HCl solution (150 ml of 0.01 N) gave an orange solid, whichwas collected by filtration, washed with H₂ O followed by Et₂ O, anddried in vacuo (25°, P₂ O₅); yield 70% (1.08 g). This material, whichwas used without further purification for conversion to 28, gave awell-resolved pmr spectrum consistent with the assigned structure. theonly extraneous signal was that due to H₂ O of hydration; pmr (DMSO-d₆),δ1.20 (m, 6, CH₃), 2.08 (m, 2, CHCH₂ CH₂), 2.40 (m, 2, CH₂ CH₂ CO), 3.46(broad s, 2, H₂ O), 4.10 (m, 4, OCH₂ CH₃), 4.44 (m, 1, NHCHCH₂), 5.30(s, 2, CH₂ OC₆ H₄), 6.74 (s, 2, NH₂), 7.16 and 7.90 (m, 4, C₆ H₄), 7.70(broad s, 2, NH₂), 8.58 (d, 1, NHCH), 8.87 (s, 1, C₇ -H). The irspectrum of the sample described is identical with that of a sampleobtained from a trial run that gave the following elemental analysisresults. Anal. Calcd for C₂₃ H₂₇ -N₇ O₆.0.5H₂ O: C, 54.54; H, 5.57; N,19.36. Found: C, 54.81; H, 5.77; N, 19.19.

EXAMPLE 28. N-[α-(2,4-diamino-6-pteridinyl)-4-anisoyl]-L-glutamic Acid(28) Monohydrate.

The diethyl ester 27 (1.00 g) was dissolved with stirring in warm DMAC(15 ml). The dark-orange solution was cooled to 25°, and NaOH solution(40 ml of 0.1 N) was added in a thin stream. Cloudiness developedinitially but soon cleared, and the solution was kept at 20°-25° underN₂ in a stoppered flask protected from light for 19 hrs. The solutionwas treated with Norit and filtered (Celite) to give a yelloworangefiltrate of pH 8.2. Careful treatment with 1 N HCl to produce pH 3.0gave a yellow precipitate. After refrigeration (3 hrs.), the mixture wascentrifuged, and the solid residue was washed twice with H₂ O (30-mlportions) with centrifugation followed by decantation. The solid wasagain suspended in H₂ O, collected by filtration, and dried in vacuo(25° over P₂ O₅ and NaOH pellets); yield 0.73 g. This sample produced athin-layer chromatogram that revealed one migrating spot that fluorescedunder uv light. A pale fluorescing spot remained at the origin. Thesample was suspended in H₂ O (5 ml) and treated with NaOH solution (11ml of 0.3 N) to redissolve. Clarification (Norit, Celite) was followedby addition of dilute HCl to pH 3.0. The precipitate was isolated asbefore and found by tlc to be homogeneous; yield 72% (0.65 g). Anal.Calcd for C₁₉ H₁₉ N₇ O₆.H₂ O: C, 49.67; H, 4.61; N, 21.34. Found: C,49.27; H, 4.23; N, 21.52. Spectral data: λ_(max), nm (ε × 10⁻³), 0.1 NHCl, 248 (32.2), 336 (11.2), 348 (sh) (10.0); pH 7, 260 (39.6), 370(7.90); 0.1 N NaOH, 260 (40.0) 370 (8.21); pmr (CF₃ CO₂ D), δ2.54 (m, 2,CHCH₂ CH₂), 2.83 (m, 2, CH₂ CO₂ H), 5.12 (m, 1, CHCO₂ H), 5.54 (s, 2,CH₂ OC₆ H₄), 7.24 and 7.94 (m, 4, C₆ H₄), 9.22 (s, 1, C₇ -H).

EXAMPLE 29. α-(2,4-Diamino-6-pteridinyl)-4-anisamide (29) Hemihydrate.

Solid NaH (127 mg of 57% dispersion in mineral oil, 3.0 mmoles) wasadded to a solution of 4-hydroxybenzamide (412 mg, 3.00 mmoles) in DMAC(15 ml). When the evolution of H₂ was complete, solid I (504 mg, 1.50mmoles) was added with stirring. After 141 hrs at about 25°, thereaction mixture was diluted with H₂ O (200 ml); the precipitate wascollected by filtration, washed with petroleum ether and dried in vacuo(78°, P₂ O₅); yield 61% (293 mg), mp >325° (Mel-Temp). This sample washomogeneous by tlc (CHCl₃ -MeOH, 5:1). Anal. Calcd for C₁₄ H₁₃ N₇O₂.0.5H₂ O: C, 52.51; H, 4.41; N, 30.61. Found: C, 52.41; H, 4.49; N,30.87. Spectral data: λ_(max), nm (ε × 10⁻³), 0.1 N HCl, 248 (25.8), 337(10.3), 347 (sh) (9.37); pH 7, 260 (29.6), 371 (6.91); 0.1 N NaOH, 260(30.0), 372 (7.55); pmr (DMSO-d₆), δ 3.36 (NH₂ and H₂ O), 5.28 (s, CH₂O), 6.72 and 7.68 (NH₂), 7.50 (m, 4, C₆ H₄), 8.86 (s, 1, C₇ -H).

EXAMPLE 30. 6-[(Phenoxy)methyl]-2,4-pteridinediamine (30).

Treatment of phenol with NaH followed by I in DMAC as described for 29led to 30, mp about 285° dec (Mel-Temp), in 70% yield (283 mg from 1.50mmoles of I) after 72 hr reaction period. (The isolation procedure wasthe same as that given for 29.). The sample was homogeneous by tlc(CHCl₃ -MeOH, 5:1) except for a faint shadow near the origin. Anal.Calcd for C₁₃ H₁₂ N₆ O: C, 58.20; H, 4.51; N, 31.33. Found: C, 57.93; H,4.26; N, 31.58. Spectral data: λ_(max), nm (ε × 10⁻³), 0.1 N HCl, 244(17.7), 276 (5.26), 287 (5.32), 337 (10.4), 347 (sh) (9.35); pH 7, 260(24.9), 372 (7.44); 0.1 N NaOH, 260 (25.1), 372 (7.58); pmr (DMSO-d₆),δ5.22 (s, 2, CH₂ O), 6.69 (s, 2, NH₂), 7.64 (s, 2, NH₂), 7.15 (m, 5, C₆H₅), 8.83 (s, 1, C₇ -H).

EXAMPLE 31. 6-[(Phenylthio)methyl]-2,4-pteridinediamine (31)Hemihydrate.

Solid I (1.50 mmoles) was added to a stirred, externally cooled (icebath) mixture of anhydrous K₂ CO₃ (221 mg, 1.60 mmoles) and thiophenol(0.5 ml, approximately 5 mmoles) in DMAC (5 ml). The mixture was stirredat 20°-25° for 21 hrs. The yellow precipitate was collected, washedsuccessively with Et₂ O and H₂ O, and dried in vacuo (78°, P₂ O₅); yield87% (370 mg), mp 270°-272° dec (Mel-Temp). The product was homogeneousby tlc (CHCl₃ -MeOH, 5:1). Anal. Calcd for C₁₃ H₁₂ N₆ S.0.5H₂ O: C,53.23; H, 4.47; N, 28.65. Found: C, 53.10; H, 4.09; N, 28.79. Spectraldata: λ_(max), nm (ε × 10⁻³), 0.1 N HCl, 247 (22.7), 342 (9.75); pH 7,260 (26.4), 285 (sh) (8.90), 375 (8.07); 0.1 N NaOH, 260 (26.9), 286(sh) (9.05), 376 (8.22); pmr (DMSO-d₆), δ 4.39 (s, 2, CH₂ S), 6.64 (s,2, NH₂), 7.42 (m, 7, C₆ H₅ and NH₂), 8.67 (s, 1, C₇ -H).

It is noted that all temperatures given herein are in degreescentigrade.

It should now be apparent that the objects initially set forth have beensuccessfully achieved. Moreover, while there is shown and describedpresent preferred embodiments of the invention, it is to be distinctlyunderstood that the invention is not limited thereto, but may beotherwise variously embodied and practiced within the scope of thefollowing claims.

ACCORDINGLY,

What we claim is:
 1. A compound having the formula of6-(bromomethyl)-2,4-diaminopteridine hydrobromide.